Project Summary/Abstract Podocyte injury is a major event in both primary glomerular disease such as focal segmental glomerulosclerosis (FSGS) and secondary glomerular disease such as diabetic kidney disease (DKD). The treatment options for these diseases are very limited. Through systems biology analysis, we identified Protein S (PS) and Tyro3 pathway as a major protective pathway in early DKD. PS binds to Tyro3, one of the TAM receptors (Tyro3, Axl, and Mer), which belong to a family of receptor tyrosine kinases that mediate anti- inflammation and cell survival. Over last several years, we have provided a large amount of data to support a podocyte protective role of PS- Tyro3 in both human and mouse glomerular disease and DKD and these findings suggest that Tyro3 could be a podocyte-protective drug target for treatment of DKD and FSGS. Therefore, we developed specific Tyro3 agonists. Among them, we found that compound 8 (C-8) and 10 (C-10) had strong activity as compared to others and therefore we selected them for further studies. We confirmed that C-10 bound directly with Tyro3 and induced its phosphorylation while it did not affect other two TAM receptors (Axl and Mer). Therefore, C-10 is a specific Tyro3 agonist. In vivo, we confirmed that C-10 attenuated proteinuria, glomerular injury, podocyte loss and foot process effacement in both Adriamycin-induced nephropathy mice and diabetic db/db mice, while these renal protective effects are lost in Tyro3 knockout mice, indicating that C-10 improves podocyte injury and glomerular disease through activation of Tyro3. Therefore, we believe that C-10 could be potentially our lead compound. These data are included in our recent paper in JCI Insights [Zhong F 2023]. In addition, our unpublished data also confirmed that C-8 had similar renal protective effects in animal models of kidney disease. Since C-8 has structural difference from C-10, C-8 could be used as our back-up compound. Based on these findings, we have applied a patent through VA (US Patent App 63-224,827 VA ID 2021-247). In this VA drug development grant, we propose the specific aims below to accelerate the development of these drugs as new therapy for DKD and FSGS, and we hope to prepare an IND application during or at the end of this application. Specific Aim 1: Characterize drug-like properties of C-10 and C-8 and determine the therapeutic window of these compounds as a novel therapy of glomerular disease. In the Aim 1.1, we will study drug metabolism and pharmacokinetics (DMPK), dose regimen, toxicity profiles of C10 and C-8. In the aim 1.2, we will further define the therapeutic window and the effective dosage of C-10 and C-8 in mouse remnant kidney model with 5/6 nephrectomy and diabetic BTBR ob/ob mice based on the DMPK data. Specific Aim 2: Build a pipeline of backup compounds for C10 and C-8 to augment drug discovery success rate. In the Aim 2.1, we will design and synthesize novel Tyro3 agonists with new structures by using pep...